Iron-sulfur clusters are present in more than 300 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. However, the most common type of cluster, the cubane-type [Fe4S4] cluster, is particularly sensitive to oxidative degradation. Hence, the process of iron-sulfur biosynthesis and repair is essential to almost all aerobic life forms and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged in bacteria, termed the NIF, ISC and SUF systems, and the ISC and SUF systems form the basis of the eukaryotic mitochondrial and plastid iron-sulfur cluster assembly machineries, respectively. In each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly, repair or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular-level understanding of iron-sulfur cluster biosynthesis and repair using the NIF, ISC and SUF systems, and accessory proteins such as monothiol glutaredoxins and thioredoxin-like Nfu proteins. Elucidating the mechanism of iron-sulfur cluster biosynthesis and repair is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload, oxidative stress and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, UV-visible absorption, circular dichroism, and magnetically-induced circular dichroism, resonance Raman, Mssbauer, and mass spectrometry) that can probe the nature, ligation and detailed properties of iron or iron-sulfur centers during cluster biosynthesis, degradation, repair or transfer to acceptor proteins. The objectives are to establish the molecular mechanisms of assembly, degradation, repair, and transfer of iron-sulfur clusters, the specificity of cluster transfer with respect to acceptor proteins, and the means by which iron-sulfur proteins regulate cellular iron homeostasis.